Cells are the basic unit of all living organisms. The one common attribute of almost all cells is that they are surrounded (or bounded) by a cytoplasmic membrane. This membrane harbors the internal contents of the cell and regulates the movement of substances into and out of the cell. Only those molecules that can diffuse across the membrane or are transported across it can move into and out of the cell. Some can pass through the lipid core of the membrane, but others must pass through pores. Still other molecules must cross the membrane attached to carriers in an energy dependent manner. Likewise, the nucleus and other cellular organelles have membranes to regulate the flow of molecules into and out of the organelle.
Fixation is a chemical process that “sets” cellular molecules in place so that the cell or tissue can then be studied. Most agents that are used as fixatives (e.g., alcohols such as ethanol and aldehydes such as paraformaldehyde) work by crosslinking cellular molecules, especially proteins. This crosslinking process prevents the degradation of the cellular structure. Various fixatives are better suited for the preservation of different cellular molecules and structures or for different detection methods. The fixative chosen for any particular purpose will be determined by the nature of that purpose.
Unfortunately, the current methods of fixation often hamper the subsequent ability of a researcher or clinician to detect internal cellular components. In other words, the very thing that prevents the degradation of the cell, fixation, can also set up a barrier to the many types of research and diagnosis that rely on larger sized detection molecules. Because of this, efforts have been made to permeabilize cells or make channels after fixation.
Current methods of permeabilizing the cell membrane after fixation are either i) not effective for all specimens, ii) are too rigorous (thus, destroying the structures to be studied) and/or iii) require expensive equipment. For example, Hoffman, et al., (U.S. Pat. No. 6,835,393) discloses the use of polycarboxylic acid polymers and pH for disrupting cell membranes but this technique can only be used on non-fixed samples. Connelly, et al., (U.S. Pat. Nos. 5,597,688 and 5,422,277) disclose the use of a composition with 2,4-dinitrobenzene sulfonic acid, 2,4-dinitrobenzoic acid or 2,4-dinitrophenol for both cell membrane fixation and permeabilization but these compositions limit the researcher's or clinician's choice of fixative and, thus, limits necessary assay flexibility. Mechanical methods such as sonication, electroporation, etc. usually only work on unfixed samples and require expensive equipment.
Furthermore, the available research and diagnostic methods of the prior art for many cellular targets such as pathologies depends on microscopic evaluations, cellular morphological parameters, staining characteristics and the presence or absence of certain targets. However, many of these diagnostic methods are not entirely accurate or sufficiently sensitive in part due to the difficulty in delivering probes to inside the cell.
Mycobacterium has a thick mycolate-rich cell wall (outer covering) which functions as an exceptionally efficient barrier. Within the cell wall is the cytoplasmic membrane. Therefore, it is very difficult to access or release nucleic acids or other cell components within the cytoplasm of the organism using standard procedures (lysis with detergents such as SDS, IGEPAL etc). Therefore, nucleic acids or internal components of the mycobacterium cell cannot be easily detected and analyzed by standard methodology such as in situ hybridization, immunofluorescence or other techniques known in the art whether the sample is from a cell culture or from patient specimen.
The one common attribute of almost all cells (including Mycobacterium sp.) is that they are surrounded (or bounded) by a cytoplasmic membrane. This membrane harbors the internal contents of the cell and regulates the movement of substances into and out of the cell. Only those molecules that can diffuse across the membrane or are transported across it can move into and out of the cell. Some molecules can pass through the lipid core of the membrane but other molecules must pass through membrane pores. Still other molecules must cross the membrane attached to carriers in an energy dependent manner. Likewise, the nucleus and other cellular organelles have membranes to regulate the flow of molecules into and out of those organelles.
What is needed are compositions and methods for the improved permeability of cell walls of Mycobacterium sp. to foreign particles such as labeled detection molecules such as nucleic acid (RNA or DNA) or peptide nucleic acid (PNA) probes with or without labeled detection moieties and methods of detecting and differentiating said Mycobacterium species. Furthermore, what is needed are compositions and methods for the improved detection of cellular targets and pathogens.